. The histidine-rich glycoprotein (HRG) of plasma has attracted attention due to its ability to interact with a variety of substances present in the circulation with physiologically relevant affinities. HRG binds metals (Zn, Ni, Cu), heme, and heparin, and associates with plasminogen, fibrinogen and thrombospondin. Notably HRG competes with antithrombin III and heparin cofactor II for heparin, and the activation of plasminogen by tissue plasminogen activator is enhanced by HRG in the presence of fibrinogen or thrombospondin. Moreover, elevated HRG levels have been observed in certain cardiovascular disorders. These activities point to a significant biological function for HRG in hemostasis; however, understanding of this function requires further study. The basic hypothesis of this research proposal is that HRG contains several ligand-binding domains that interact with a variety of ligands of diverse structure and thereby enable HRG to accomplish its role in modulating hemostasis. A prerequisite for elucidation of the mechanisms of action of HRG in hemostasis in vivo is that the activity and structure of HRG and of each of its several domains he defined. A corollary to this hypothesis is that the unique structure of HRG is a major key to understanding how it functions. This hypothesis is supported by our isolation of active fragments of HRG (80 kDa): an N-terminal domain (app. Mr 45 kDa which interacts with plasminogen; a unique histidine-proline-rich domain (app. Mr28kDa) which binds heparin and metals; and a C-terminal domain (app. Mr16kDa) which also binds heparin. An extension of this hypothesis is that the circulating levels of HRG will influence its effects on hemostasis, so that information on the regulation of HRG levels must ultimately be sought. The specific aims here are to: (1) complete definition of the biochemical bases of the interaction of HRG with heparin, plasmin(ogen), fibrinogen and thrombospondin; (2) continue to characterize the structure, conformation and ligand binding activity of the functional domains of HRG and the interrelationships among these binding domains; (3) extend study of the mechanisms of the modulating effects of HRG on the activation of plasminogen and on plasmin activity; and (4) complete the elucidation of the three-dimensional structure of HRG. The goal of this proposal is the elucidation of the biochemical mechanisms of the biological activities of HRG. The approach taken here is expected to achieve this goal and provide information of importance not only in establishing the mechanisms of action of HRG in vivo but also in the development of diagnostic tools for, and therapeutic approaches to, hemostatic disorders involving HRG.